Identification DEGs of FSGS and MCD
We identified 105 glomerular samples, comprising 48 FSGS samples, 30 MCD and 27 normal samples, in the GSE104948 and GSE108113 datasets. Based on the cut-of criteria of adj P-value ≤ 0.05,
A total of and 358 and 368 glomerular genes were significantly differentially expressed in FSGS and MCD compared with healthy controls. Of note, A total of 156 overlapping DEGs were identified in FSGS were also differentially expressed in MCD. There were 202 and 212 specific DEGs remained for FSGS and MCD, respectively. The complete lists of shared DEGs and the remaining DEGs were presented in (Fig. 1A). As shown in volcano plot 223 DEGs were upregulated and 135 DEGs were downregulated in FSGS, 193 DEGs were upregulated and 175 DEGs were downregulated in MCD. The results of the expression level analysis are presented in a volcano plot in Figure.1B and C. As indicated in the clustering heat map (Fig. 1B C), these DEGs could well distinguish the FSGS and MCD vs healthy control.
GO and pathway enrichment analysis of DEGs
To further perform a systematic characterization and explore the biological functions of the DEGs in FSGS and MCD, functional annotation and pathway analyses, including GO and KEGG analyses, were performed using DAVID. GO analysis showed that most of the biological processes (BP) terms of the DEGs in these two diseases were simultaneously enriched in mRNA splicing, RNA polymerase II transcription, mRNA export, insulin stimulus, integrin-mediated signaling pathway, viral process and phagocytosis. while some BP terms involved in immune response, autophosphorylation and actin cytoskeleton organization were specific enriched in FSGS. DNA methylation and apoptotic process were only enriched in MCD.(Fig. 2)
CC terms such as focal adhesion, catalytic step 2 spliceosome, nucleus, spliceosomal complex were enriched in both diseases. CC terms of extrinsic component of cytoplasmic side of plasma membrane, AIM2 inflammasome complex and endoplasmic reticulum membrane only enriched in FSGS. Terms of extracellular exosome, dendritic spine, axonal growth cone and cytoplasmic vesicle only enriched in MCD. As for MF term, we also found out some terms related to RNA, nucleotide and protein binding enriched in these two diseases at the same time. While tyrosine kinase, cysteine-type endopeptidase and aminoacyl-tRNA ligase activity only enriched in FSGS. Some MF terms associated with specific chromatin, protein and domain binding, protein kinase and transcription coactivator activity were specific in MCD. (Fig. 2)
KEGG pathway analysis indicated that the DEGs both in FSGS and MCD were enriched in Spliceosome, Vibrio cholerae infection and FoxO signaling pathway simultaneously (Fig. 3.C). In addition, certain KEGG pathways, include Platelet activation, Osteoclast differentiation, Fc gamma R-mediated phagocytosis, Renin secretion, Fc epsilon RI signaling pathway, Insulin resistance and Pathogenic Escherichia coli infection were commonly involved in the development of FSGS. (Fig. 3A) Some pathway such as Apoptosis, Estrogen signaling pathway, Insulin signaling pathway and Sphingolipid signaling pathway were involved in MCD exclusively. (Fig. 3B)
PPI Network Construction and Module Analysis
The PPI network of the DEGs was constructed and the most significant module was obtained using MCODE plugin of Cytoscape (Fig. 4). GO analysis showed that most of the BP terms in these the top module of FSGS, MCD and overlapped DEGs were mainly enriched in mRNA splicing, RNA mRNA export and termination (Fig. 5). KEGG pathway analysis showed that among these three modules, KEGG pathway only enriched in Spliceosome which means MCD and FSGS shared the same pathogenic pathways, this is also can explain why they have the similar clinical manifestations and pathologic changes.
Identification and analysis of hub genes
We exported the STRING data to Cytoscape to construct and visualize the PPI network by implementing cytoHubba. Thereafter, we implemented the MCC method to evaluate the significance of the genes in the network. There are seven same hub genes shared both in FSGS and MCD, SR splicing-factor 5 (SRSF5), Heterogeneous Nuclear Ribonucleoprotein A2/B1(HNRNPA2B1), Crooked Neck Pre-MRNA Splicing Factor 1(CRNKL1), DEAD-Box Helicase 5(DDX5), Cleavage Stimulation Factor Subunit 2 Tau Variant (CSTF2T), Splicing Factor 3b Subunit 1(SF3B1)and Serine And Arginine Rich Splicing Factor 7(SRSF7).while Splicing factor 3A, subunit 3(SF3A3), DExD-Box Helicase 39A( DDX39A) and RNA Binding Motif Protein 25 ( RBM25) only as hub genes in MCD, CD2BP2, LSM8 and SNRPB as hub genes in FSGS. Further analysis found out that, among these hug genes, CD2BP2, LSM8 and SNRPB only differential expression in FSGS and SF3A3 only differential expression in MCD, which may be used for differential diagnosis of these two diseases in the future(Fig. 6).